The proposed study is designed to prove that highly oriented enamel-like nano- and micro-structures of acid-etched human enamel can be generated using pyrophosphate (PPi)-stabilized highly supersaturated solutions in the presence of specific molecules that exhibit the capacity to guide the epitaxial growth of mature enamel crystals. This study is guided by recent advances in the PI's laboratory that have provided unique insight into the mechanism by which enamel matrix proteins regulate enamel formation and strong preliminary data that support the feasibility of proposed biomimetic approaches for enamel regeneration. Despite tremendous efforts in promoting oral hygiene and fluoridation, further research is needed to achieve an easy-to-apply, fast growing enamel-like bioceramic for biomimetic repair. Thus, there is a great need to develop effective means to regenerate tooth structures. The central hypothesis is that the restoration of proper enamel structure and function can be achieved through the regulation of mineral ion availability, crystal growth kinetics, and crystal orientation. Long-term, a better understanding of the mechanism of enamel mineral formation will aid in the development of novel biomimetic and biocompatible restorative materials for enamel regeneration and, for example, the treatment of early dental caries. Amelogenin-like materials, especially key functional sequences of amelogenin retained within commercially synthesized leucine-rich amelogenin peptide (LRAP) can potentially be used in the regeneration of tooth enamel structure and properties. The goals of this proposal will be achieved through the completion of the following two Specific Aims: Aim 1. To determine the mechanism and effectiveness of LRAP and non-phosphorylated full-length amelogenin to guide the regeneration of enamel structure in vitro using pyrophosphate (PPi)-stabilized supersaturated calcium phosphate solutions. Mineralization kinetics and epitaxial growth of acid-etched human enamel will be regulated by the hydrolysis of the PPi mineralization inhibitor by 1) the enamel surface itself and 2) alkaline phosphatase (AP). Aim 2. To determine the mechanism and effectiveness of highly supersaturated calcium phosphate solutions that are stabilized by phosphorylated native and synthetic amelogenins using AP to trigger the regeneration of the enamel structure in vitro. Full-length native (phosphorylated) porcine amelogenin and LRAP(+P), potent stabilizers of supersaturated calcium phosphate solutions, will be studied as substitutes for PPi and examined also for their additional potential to guide th regeneration of the acid-etched enamel mineral structure upon dephosphorylation by added AP. The extent, nature, and orientation of formed mineral will be assessed using SEM, EDX, FT-IR and grazing incidence X-ray diffraction. The restoration and/or improvement of base-line enamel properties will be assessed with respect to mechanical properties, physico- chemical properties, and strength of mineral attachment.